1. Field of the Invention
2. General Background and State of the Art
This invention is directed to a bone graft material incorporating demineralized bone matrix and lipids for particular use in enhancing bone formation.
One of the few tissues that regenerates in mammals is bone. To a great extent, this is due to the ability of specific growth factors to stimulate stem cells along the chondrogenic and osteogenic pathways and the role of mechanical forces that encourage bone remodeling.
Significant efforts have been made to enhance bone healing using decalcified bone matrix as an inducer. Decalcified bone matrix, which is mostly collagen with small amounts of growth- and differentiation-inducing molecules, is able to stimulate bone formation, even after implantation (subcutaneously or intramuscularly) at ectopic sites where there is no bone. The chondro-osteogenic response induced by implants of demineralized rabbit (M. R. Urist and T. A. Dowell, xe2x80x9cThe Inductive Substratum for Osteogenesis in Pellets of Particulate Bone Matrix,xe2x80x9d Clin. Orthoped. Rel. Res. 61:61-68 (1969); M. R. Urist and B. S. Strates, xe2x80x9cBone Morphogenetic Protein,xe2x80x9d J. Dent. Res. 50:1392-1406 (1971)) and rat bone matrix (C. B. Huggins et al., xe2x80x9cTransformation of Fibroblasts by Allogeneic and Xenogeneic Transplants of Demineralized Tooth arid Bone,xe2x80x9d J. Exp. Med. 132:1250-1258 (1970); A. H. Reddi and C. B. Huggins, xe2x80x9cInfluence of Transplanted Tooth and Bone on Transformation of Fibroblasts,xe2x80x9d Proc. Soc. Exp. Biol. Med. 143:634-637 (1973); G. D. Syftestad and M. R. Urist, xe2x80x9cDegradation of Bone Matrix Morphogenetic Activity by Pulverization,xe2x80x9d Clin. Orthoped. Rel. Res. 141:281-286 (1979)).
The data suggested that bone morphogenetic protein was associated with a relatively acid-resistant, trypsin labile, water-insoluble non-collagenous protein of previously uncharacterized nature (B. S. Strates and J. J. Tideman, xe2x80x9cContribution of Osteoinductive and Osteoconductive Properties of Demineralized Bone Matrix to Skeletal Repair,xe2x80x9d Euro. J. Exp. Musculoskel. Res. 2:61-67 (1993); M. R. Urist et al., xe2x80x9cSolubilized and Insolubilized Bone Morphogenetic Protein,xe2x80x9d Proc. Natl. Acad. Sci. USA 76:1828-1832 (1979). This has been followed by extensive work attempting to further purify factors with bone morphogenetic potential (M. R. Urist, xe2x80x9cThe Search for Discovery of Bone Morphogenetic Protein (BMP),xe2x80x9d In Bone Grafts, Derivatives, and Substitutes (M. R. Urist, B. T. O""Conner and R. G. Burwell, eds., Butterworth Heineman, London, 1994), pp. 315-362), and subsequently to generate active recombinant molecules of human origin.
The most widely used carrier for BMPs, either as a mixture of the bioactive factor extracted directly from human or animal bones using procedures described in the literature or recombinant molecules of the most active individual species, is DBM (demineralized bone matrix) from cortical bone of various animal sources. It is purified by a variety of procedures for the removal of non-collagenous proteins and other antigenic determinants. It usually consists of more than 99% type I collagen. Essentially, one can look at this approach as one of restoring an otherwise inactive bone matrix to an active form, as previously described.
Acid (HCl)-demineralized bone matrix, which contains a mixture of BMPs, consistently induces formation of new bone with a quantity of powdered matrices in the 10-25 mg range, while less than 10 mg fails to induce bone formation. Addition of acid-soluble type I collagen and chondroitin-6-sulfate promoted bone yield. Bone matrix inactivated by extraction with guanidine hydrochloride, an efficient protein denaturant, restores bone-forming activity by the addition of partially purified BMP-3 or recombinant BMP-2. Thus, new bone formation requires a combination of BMP and an insoluble collagenous substratum that can include chondroitin-6-sulfate (M. Muthukumaran et al., xe2x80x9cDose-Dependence of and Threshold for Optimal Bone Induction by Collagenous Bone Matrix and Osteogenin-Enriched Fraction,xe2x80x9d Col. Rel. Res. 8:433-441 (1988); R. G. Hammonds, Jr. et al, xe2x80x9cBone-Inducing Activity of Mature BMP-2b Produced from a Hybrid BMP-2a/2b Precursor,xe2x80x9d Mol. Endocrinol. 5:149-155 (1991); U. Ripamonti et al., xe2x80x9cThe Critical Role of Geometry of Porous Hydroxyapatite Delivery System in Induction of Bone by Osteogenin, a Bone Morphogenetic Protein,xe2x80x9d Matrix 12:202-212 (1992); U. Ripamonti et al., xe2x80x9cInduction of Bone in Composites of Osteogenin and Porous Hydroxyapatite in Baboons,xe2x80x9d Plast. Reconstr. Surg. 89:731-739 (1992)). The same preparation of partially purified bovine BMP-3 was inactive when implanted subcutaneously without an insoluble collagenous matrix, but when combined with collagen, induced bone formation.
Reconstituted collagen can sometimes be used as a carrier for BMPs with less predictable results. However, a fibrous form of purified telopeptide-free collagen, which is intrinsically less soluble and biodegradable than the reconstituted collagen, is an effective carrier material.
Lipids were found to be present in very large amounts at various sites of normal bone formation (J. T. Irving and R. E. Wuthier, xe2x80x9cHistochemistry and Biochemistry of Calcification with Special Reference to the Role of Lipids,xe2x80x9d Clin. Orthoped. Rel. Res. 56:237-260 (1968). Although there have been suggestions that the incidence and quantity of bone formation are greatest when BMP is delivered combined with the tissue using various biological synthetic materials (M. R. Urist et al., xe2x80x9cLipids Associated Closely with Bone Morphogenetic Protein (BMP) and Induced Heterotopic Bone Formation,xe2x80x9d Connect. Tissue Res. 36:9-20 (1997)), there is still a need for improved carriers for optimal results. There has been no suggestion that lecithin would serve as a suitable carrier. In work reported by Urist and coworkers (M. R. Urist et al., xe2x80x9cNeutral Lipids Facilitate Transfer of Bone Morphogenetic Proteins and Other Noncollagenous Proteins,xe2x80x9d Med. Hypotheses 49 :465-475 (1997), composites of recombinant BMP-2 and acetone-soluble lipids were reported to induce larger deposits of bone than implants of recombinant BMP-2 without acetone soluble lipids. Acetone soluble lipids consisted chiefly of triglycerides, cholesterol, and saturated short chain fatty acids, and included little or no phospholipids.
There is particularly a need for an improved matrix for supplying the demineralized bone matrix that is easily moldable, biocompatible, slowly resorbable, and insoluble in tissue fluids. There is a particular need for the development of compositions that can be varied in their physical form and consistency so that they can be made more solid or more liquid as the need requires.
In general, a demineralized bone putty composition according to the present invention comprises:
(1) demineralized bone matrix (DBM); and
(2) a lipid fraction selected from the group consisting of lecithin and a mixture of lecithin and triglycerides containing unsaturated fatty acids.
The mixture is such that the putty composition is moldable, biocompatible, slowly resorbable, insoluble in tissue fluid, and non-extrudable.
Typically, the composition comprises about 30-40% of DBM and about 60% of the lipid fraction.
In one embodiment of the present invention, the lipid fraction comprises lecithin.
In another embodiment of the present invention, the lipid fraction comprises a mixture of lecithin and triglycerides containing unsaturated fatty acids. Typically, when the composition contains a mixture of lecithin and triglycerides, the mixture of lecithin and triglycerides containing unsaturated fatty acids is about a 1:1 mixture.
The composition can further comprise a long-chain saturated fatty acid such as palmitic acid.
The DBM can be human DBM, rat DBM, or DBM from another animal such as a cow, a horse, a pig, a dog, a cat, a sheep, or another socially or economically important animal species. In one preferred embodiment, the DBM is delipidated, such as by treatment with a chloroform-methanol mixture.
The composition can further comprise other ingredients. In one alternative, the composition can further comprise a calcium salt.
In another alternative, the composition can further comprise at least one antioxidant selected from the group consisting of Vitamin E and Vitamin C.
In still another alternative, the composition can further comprise an ingredient selected from the group consisting of methylcellulose and hydroxypropyl methylcellulose.
The lipid fraction of the composition can be sterilized by radiation using standard radiation sterilization techniques.
The composition can be formulated so that the phospholipids are solid or liquid at room temperature.